IMMUNOASSAY FOR SARS-CoV-2 ANTIBODIES

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the strain of coronavirus that causes coronavirus disease 2019 (COVID-19), the respiratory illness responsible for the COVID-19 pandemic. Antibodies produced from an immune response against SARS-CoV-2 infection are used to analyze prior exposure to the virus. The present invention provides methods for detecting antibodies in response to SARS-CoV-2 infection in a single multiplex immunoassay.

FIELD OF THE INVENTION

The present invention relates generally to multiplex immunoassays andspecifically to the detection of SARS-CoV-2 antibodies produced inresponse to a SARS-CoV-2 infection.

BACKGROUND INFORMATION

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is thestrain of coronavirus that causes coronavirus disease 2019 (COVID-19),the respiratory illness responsible for the COVID-19 pandemic.SARS-CoV-2 is an IV positive-sense single-stranded RNA virus that iscontagious in humans.

Each SARS-CoV-2 virion is 50-200 nanometers in diameter. Like othercoronaviruses, SARS-CoV-2 has four structural proteins, known as the SP(spike), E (envelope), M (membrane), and NP (nucleocapsid) proteins; theNP protein holds the RNA genome, and the SP, E, and M proteins togethercreate the viral envelope. The spike protein, which has been imaged atthe atomic level using cryogenic electron microscopy, is the proteinresponsible for allowing the virus to attach to and fuse with themembrane of a host cell; specifically, its S1 subunit catalyzesattachment, the S2 subunit fusion.

Protein modeling experiments on the spike protein of the virus soonsuggested that SARS-CoV-2 has sufficient affinity to the receptorangiotensin converting enzyme 2 (ACE2) on human cells to use them as amechanism of cell entry. It has been shown that ACE2 could act as thereceptor for SARS-CoV-2. Studies have shown that SARS-CoV-2 has a higheraffinity to human ACE2 than the original SARS virus strain. SARS-CoV-2may also use the protein basigin (CD147) to assist in cell entry.

Initial SP priming by transmembrane protease, serine 2 (TMPRSS2) isessential for entry of SARS-CoV-2. After a SARS-CoV-2 virion attaches toa target cell, the cell's protease TMPRSS2 cuts open the SP of thevirus, exposing a fusion peptide in the S2 subunit, and the hostreceptor ACE2. After fusion, an endosome forms around the virion,separating it from the rest of the host cell. The virion escapes whenthe pH of the endosome drops or when cathepsin, a host cysteineprotease, cleaves it. The virion then releases RNA into the cell andforces the cell to produce and disseminate copies of the virus, whichinfect more cells. SARS-CoV-2 produces at least three virulence factorsthat promote shedding of new virions from host cells and inhibit immuneresponse.

There is a need for a rapid and accurate diagnostic test for thedetection of a SARS-CoV-2 infection. Ideally, the diagnostic test woulddetect evidence of a prior infection, e.g., antibodies produced againstthe SARS-CoV-2 virus.

SUMMARY OF THE INVENTION

The present invention is based on the seminal discovery of the use ofmultiplex immunoassays for detection of infection caused by SARS-CoV-2,e.g., COVID-19. Specifically, the invention provides immunoassays thatdetect antibodies produced in response to infection by SARS-CoV-2.

In one embodiment, the present invention provides, a substrate with atleast two capture elements specific for SARS-CoV-2 on the substrate,each capture element corresponding to and being able to bind a targetanalyte, the substrate further optionally with a plurality of controlelements comprising: at least one fiduciary marker, at least onenegative control to monitor background signal, at least one negativecontrol to monitor assay specificity, at least one positive colorimetriccontrol, at least one positive control to monitor assay performance andany combination thereof. In one aspect, the capture elements bind targetanalytes, wherein the target analytes are indicative of exposure toSARS-CoV-2 and/or COVID-19. In one aspect, the target analyte is anantibody, an antibody fragment, an antibody binding domain, or anycombination thereof. In another aspect, the target analyte is aSARS-CoV-2 antibody, fragment or binding domain thereof In an additionalaspect, the capture element is a protein, a protein fragment, a bindingprotein (BP), a binding protein fragment, an antigen, a virus protein,or any combination thereof. In one aspect, the capture element is avirus structural protein or epitope thereof. In an additional aspect,the virus structural protein or epitope thereof is selected from aSARS-CoV-2 Membrane protein (MP), Nucleocapsid protein (NP), Spikeprotein (SP), fragment thereof or any combination thereof In a furtheraspect, the virus structural protein or epitope thereof is aNucleocapsid protein or Nucleocapsid protein fragment. In one aspect,the substrate is a solid or a porous substrate. In an additional aspect,the solid substrate is a paramagnetic bead, microtiter plate,microparticle, or a magnetic bead. In another aspect, the poroussubstrate is a membrane.

In an additional embodiment, the present invention provides a kit fordetecting a plurality of target analytes in a sample, containing asubstrate and optionally one or both of a background reducing reagent,and a colorimetric detection system. In one aspect, the kit alsocontains one or more items from a wash solution, one or more antibodiesfor detection of antigens, ligands or antibodies bound to the captureelements or for detection of the positive controls, software foranalyzing captured target analytes, and a protocol for measuring thepresence of target analytes in samples. In an additional aspect, theantibodies for detection are antibody-binding protein (BP) conjugates,antibody-enzyme label conjugates, or any combination thereof. In afurther aspect, the sample is a nasal swab or a blood sample, e.g.,serum and/or plasma. In one aspect, the substrate is a solid or a poroussubstrate. In an additional aspect, the solid substrate is aparamagnetic bead, microtiter plate, microparticle, or a magnetic bead.In another aspect, the porous substrate is a membrane.

In a further embodiment, the present invention provides methods ofdetecting exposure of a subject to SARS-CoV-2 by contacting a substratewith a biological sample from the subject, wherein the subject issuspected of having COVID-19 or at risk of having COVID-19; anddetecting the presence of an antibody that binds to SARS-CoV-2, or acombination thereof, thereby detecting exposure of the subject toSARS-CoV-2. In one aspect, the detection method is a colorimetric,absorbance, chemiluminescence or a fluorescence signal. In certainaspects, the detection method is electrochemical, surface plasmonresonance, localized surface plasmon resonance or interferometry. In anadditional aspect, the antibody is IgG and/or IgM. In a further aspect,the sample is a blood sample, e.g., serum and/or plasma.

In another embodiment, the present invention provides methods forprocessing a microarray by providing a substrate, adding at least onesample to the substrate, and processing the substrate such that adetectable result is given by two or more of at least one fiduciarymarker, at least one positive colorimetric control, and at least onepositive control to monitor assay performance.

In one embodiment, the present invention provides methods for detectingan analyte in a sample comprising providing a substrate, adding at leastone sample to the substrate, and processing the substrate such that adetectable result is provided. In one aspect, the detectable resultincludes two or more of at least one fiduciary marker, at least onepositive colorimetric control, and at least one positive control todetect an analyte in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an overview of the COVID-19 multiplex immunoassay (MIA).Indirect immunoassay (IA) for the detection of antibodies (IgM and IgG)against SARS-CoV-2.

FIGS. 2A-E show a COVID-19 MIA protocol design. FIG. 2A. Printing ofSARS-CoV-2 structural proteins on the assay surface. FIG. 2B. Blockingof assay surface after protein/Ab printing. FIG. 2C. Detection of targetCOVID-19 analytes in the patient sample. FIG. 2D. Detection ofspecifically bound COVID-19 analytes by binding with HRP-labelleddetection Ab against the analytes. FIG. 2E. Generation of colorimetricarray spots by the addition of HRP substrate (TMB).

FIGS. 3A-B show a COVID-19 MIA—Membrane-based, Single well assay. FIG.3A. 16-well platform employing the nitrocellulose membrane as thesubstrate for the printing of spots. FIG. 3B. capture antibody againstNP and the SARS-CoV-2 structural proteins, i.e. NP, MP, and SP, areprinted in duplicate. The positive control spots are printed in thewell. The white circles signify that nothing has been printed at thatspecific position.

FIGS. 4A-B show a COVID-19 MIA—Membrane-based, Single well, usinggrouped SARS-CoV-2 structural protein assay. FIG. 4A. 16-well platformemploying the nitrocellulose membrane as the substrate for the printingof spots. FIG. 4B. Grouped SARS-CoV-2 structural proteins, i.e. NP, MP,and SP, in a mixture are printed in duplicate in the same well. Thepositive control spots are printed in the well. The white circlessignify that nothing has been printed at that specific position.

FIGS. 5A-B show a COVID-19 MIA—Membrane-free, Single well assay. FIG.5A. 96-well microtiter plate (12 detachable strips of 8 wells each) isused as substrate for the printing of spots. FIG. 5B. Capture Ab againstNP and the SARS-CoV-2 structural proteins, i.e. NP, MP, and SP, areprinted in duplicate in each well of another strip. The positive controlspots are printed in the well. The white circles signify that nothinghas been printed at that specific position.

FIGS. 6A-B show a COVID-19 MIA—Membrane-free, Single well, using groupedSARS-CoV-2 structural proteins assay. FIG. 6A. 96-well microtiter plate(12 detachable strips of 8 wells each) is used as substrate for theprinting of spots. FIG. 6B. Grouped SARS-CoV-2 structural proteins, i.e.NP, MP, and SP, in a mixture are printed in duplicate in each well ofanother strip. The positive control spots are printed in all the wells.The white circles signify that nothing has been printed at that specificposition.

FIGS. 7A-B show COVID-19 MIA (Membrane-based, Single well assay). FIG.7A. 16-well platform employing the nitrocellulose membrane as thesubstrate for the printing of spots. FIG. 7B. The SARS-CoV-2 structuralproteins, i.e., NP and SP, are printed in duplicates.

FIG. 8 shows an assessment of First WHO International Reference Panelfor anti-SARS-CoV-2 Immunoglobulin using the COVID-19 MIA.

FIGS. 9A-B show COVID-19 multiplex immunoassay ELISA-basedformat—conventional 96 wells ELISA plate (12 strips of 8 wellseach)-membrane free. FIG. 9A. Conventional 96 wells ELISA plate. FIG.9B. NP and SP SARS-CoV-2 proteins printed in duplicate.

FIG. 10 shows Wells' regions used to calculate the assay background. Theassay background was calculated as a median of the intensity obtainedacross the region inside the dark circles.

FIGS. 11A-E show the printing layout and wells at the end of an assay.FIG. 11A. Printing layout. FIG. 11B. Assay run using Anti-N Proteinreconstructed human mAb, IgG at 1 μg/ml as a sample. FIG. 11C. Assay runusing Anti-Spike-RBD human reconstructed mAb, IgG at 1 μg/ml as asample. FIG. 11D. Assay run using a sample (Panel #18) which is reactivefor both SARS-CoV-2 Nucleocapsid Protein and SARS-CoV-2 SpikeGlycoprotein (51). FIG. 11E. Assay run using a sample (Panel #34) whichis non-reactive for both printed antigens. The visible signal atSARS-CoV-2 NP spots is the highest background observed across all thenon-reactive samples tested.

FIG. 12 shows an assessment of First WHO International Reference Panelfor anti-SARS-CoV-2 Immunoglobulin using the COVID-19 MIA—membrane free,single well format.

FIGS. 13A-B show COVID-19 multiplex immunoassay ELISA-based format—96wells ELISA plate (12 strips of 8 wells each)-membrane free. FIG. 13A.Conventional 96 wells ELISA plate. FIG. 13B. NP and SP SARS-CoV-2proteins are mixed then printed in duplicate.

FIG. 14 shows the Wells' regions used to calculate the assay background.The assay background was calculated as a median of the intensityobtained across the region inside the dark circles.

FIGS. 15A-E show printing layout and wells at the end of an assay. FIG.15A. Printing layout. FIG. 15B. Assay run using Anti-N Proteinreconstructed human mAb, IgG at 1 μg/ml as sample. FIG. 15C. Assay runusing Anti-Spike-RBD human reconstructed mAb, IgG at 1 μg/ml as sample.FIG. 15D. Assay run using a sample (Panel #18) which is reactive forboth SARS-CoV-2 Nucleocapsid Protein and SARS-CoV-2 Spike Glycoprotein(51). FIG. 15E. Assay run using a sample (Panel #34) which isnon-reactive for both printed antigens. The visible signal at SARS-CoV-2NP&SP mixed spots is the highest background observed across all thenon-reactive samples tested.

FIG. 16 shows an assessment of First WHO International Reference Panelfor anti-SARS-CoV-2 Immunoglobulin using the COVID-19 MIA—membrane free,single well, using grouped SARS-CoV-2 structural proteins format.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the seminal discovery of the use ofmultiplex immunoassays for detection of antibodies produced in responseto infection by SARS-CoV-2.

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to particularcompositions, methods, and experimental conditions described, as suchcompositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyin the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein, which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, it will be understood thatmodifications and variations are encompassed within the spirit and scopeof the instant disclosure. The preferred methods and materials are nowdescribed.

The present invention enables the in vitro diagnosis of COVID-19 via amultiplex immune assay (MIA) that detects antibodies produced againstSARS-CoV-2 infection (e.g., IgG and IgM). Therefore, COVID-19 isdiagnosed at a very early-stage starting from about 3 days from theonset of infection when nucleocapsid protein (NP) is shed in patients.The peak levels of NP have been observed in humans at about 10 daysafter onset of infection, which continuously decreases in the patientsand becomes undetectable. The seroconversion of antibodies (IgG, IgM andIgA) against SARS-CoV-2 has been shown to occur between about 16-23 daysfrom the onset of infection.

In one embodiment, the present invention provides, a substrate with atleast two capture elements specific for SARS-CoV-2 on the substrate,each capture element corresponding to and being able to bind a targetanalyte, the substrate further optionally with a plurality of controlelements comprising at least one fiduciary marker, at least one negativecontrol to monitor background signal, at least one negative control tomonitor assay specificity, at least one positive colorimetric control,at least one positive control to monitor assay performance and anycombination thereof. In one aspect, the capture elements bind targetanalytes, wherein the target analytes are indicative of COVID-19. Inanother aspect, the target analyte is a SARS-CoV-2 antibody, fragment orbinding domain thereof In an additional aspect, the capture element is aprotein, a protein fragment, a binding protein (BP), a binding proteinfragment, an antigen, an antigenic determinant, a virus protein, or anycombination thereof In one aspect, the capture element is a virusstructural protein or epitope thereof In an additional aspect, the virusstructural protein or epitope thereof is selected from a SARS-CoV-2Membrane protein (MP), Nucleocapsid protein (NP), Spike protein (SP), orany combination thereof. In a further aspect, the virus structuralprotein or epitope thereof is a Nucleocapsid protein or Nucleocapsidprotein fragment. In one aspect, the substrate is a solid or a poroussubstrate. In an additional aspect, the solid substrate is aparamagnetic bead, microtiter plate, microparticle, or a magnetic bead.In another aspect, the porous substrate is a membrane.

As used herein, the term “substrate” is any surface that supports animmunoassay. The substrate of the invention may be a solid substrate ora porous substrate, for example.

In certain aspects, the substrate is a solid substrate. Examples ofsolid substrates include, but are not limited to, 96 well microtiterplate, glass, microbeads, nano/micro-particles and magnetic beads. Inone aspect, a 96 well microtiter plate is polystyrene, PDMS, PMMA,polycarbonate, cyclic polyolefins, Zeonor, Zeonex, or cellulose acetate.In various aspects, the solid substrate maybe glass beads,nano-/microparticles, magnetic beads or paramagnetic beads.

In some aspects, the porous substrate is a membrane. The term “porousmembrane” refers to a membrane with protein binding characteristics anda narrow pore-size distribution (e.g,. microporous). In one embodiment,the porosity of the membrane may determine the exposure time of reagentswith membrane bound components by controlling the flow rate through themembrane. Microporous membranes for use in the present invention includeby way of example, nitrocellulose, nylon, polyvinylidene difluoride,polyester, polystyrene, polyethersulfone, cellulose acetate, mixedcellulose esters and polycarbonate. For example, PictArray™ (U.S. Pat.No. 9,625,453)

The choice of membrane is typically dependent on three main membranecharacteristics: protein-binding capacity, porosity, and strength. Theability of the membrane to immobilize macromolecules, in particularproteins, is important as the membrane serves as the solid phase used inthe assay. However, this ability must be balanced with the availabilityof appropriate reagents (for example, blockers) for blockingnon-specific interactions on the membrane. Similarly, in a flow-throughconfiguration, the porosity of the membrane may determine the exposuretime of reagents with membrane bound components by controlling theirflow rate through the membrane. However, porosity must be balanced withthe degree of array spot spreading during array manufacture, which canresult in decreased signal intensity or cross contamination betweenadjacent spots. The strength of the membrane is important for themanufacture and eventual use of a device. A wide range of membranes areavailable with differing characteristics, allowing a particular membraneto be chosen depending on the requirements of an assay.

In preferred embodiments, microporous membranes for use in the presentinvention comprise nitrocellulose, nylon, polyvinylidene difluoride,polyester, polystyrene, polyethersulfone, cellulose acetate, mixedcellulose esters and polycarbonate.

While some membranes such as cellulose acetate may have insufficientbinding capacities for diagnostic immunoassays, the characteristics ofsuch membranes may be applicable for assays where lower levels ofaccuracy or sensitivity are sufficient.

The microporous membrane is removably attachable to a bottomlessmicrotiter plate for example. Accordingly, the membrane can be dividedinto individual microtiter wells that are separated from each other by aphysical barrier, to prevent sample mixing between wells. Moreover,different assays can be conducted in separate wells, requiring smallervolumes of assay reagents.

The assay elements (control and capture elements) are placed on thesubstrate surface, with or without an adapter molecule between themembrane and the element. Preferably, the assay elements bind to thesubstrate by covalent or non-covalent interaction. One of skill in theart will recognize that methods of placing assay elements on thesubstrate include printing, spotting or other techniques known in theart. For purposes of the present application, the term “printing” can beused to include any of the methods for placing the assay elements on amembrane.

The terms “array” or “microarray” as used herein refer to a collectionof multiple assay elements on a substrate. Specifically, an array is acollection of capture elements and/or control elements on a substrate.

In various aspects, the elements on the array are placed on thesubstrate in discrete areas of between 100 μm to 500 μm in diameter.More preferably, the discrete areas are between 350 μm to 400 μm indiameter. In certain aspects, the discrete areas of the array are placedin a 5×5 grid. In one aspect, the array comprises up to nine controlelements and two replicates of each of eight different capture elements.In one aspect, the capture elements are printed in two or morereplicates of four different capture elements and multiples thereof.

As used herein, the term “assay element” refers to any of a number ofdifferent elements for use in an array of the invention. Exemplary assayelements include, but are not limited to, capture elements and controlelements.

The term “capture element” refers to a molecule that is able to bind toa target analyte. Examples of useful capture elements include proteins,protein fragments, polypeptides, polypeptide fragments, bindingproteins, binding protein fragments, antibodies (polyclonal, monoclonal,or chimeric), antibody fragments, antibody heavy chains, antibody lightchains, single chain antibodies, single-domain antibodies (a VHH forexample), Fab antibody fragments, Fc antibody fragments, Fv antibodyfragments, F(ab′)2 antibody fragments, Fab′ antibody fragments,single-chain Fv (scFv) antibody fragments, antibody binding domains,antigens, antigenic determinants, epitopes, haptens, immunogens,immunogen fragments, binding domains, a metal ion, a metal ion-coatedmolecule, biotin, avidins, streptavidins; substrates, enzymes, abzymes,co-factors, receptors, receptor fragments, receptor subunits, receptorsubunit fragments, ligands, inhibitors, hormones, binding sites,lectins, polyhistidines, coupling domains, oligonucleotides, and a virusprotein. Useful capture elements will correspond to and be able to binda specific target analyte, such as a molecule or class of molecules thatare present in a sample to be tested.

In one embodiment, the capture element is selected from a protein, aprotein fragment, a binding protein, a binding protein fragment, anantibody, an antibody fragment, an antibody heavy chain, an antibodylight chain, a single chain antibody, a single-domain antibody (a VHHfor example), a Fab antibody fragment, an Fc antibody fragment, an Fvantibody fragment, a F(ab′)2 antibody fragment, a Fab′ antibodyfragment, a single-chain Fv (scFv) antibody fragment, an antibodybinding domain, an antigen, an antigenic determinant, an epitope, ahapten, an immunogen, an immunogen fragment, a binding domain; metalion, or metal ion-coated molecule, biotin, avidin, streptavidin; asubstrate, an enzyme, an abzyme, a co-factor, a receptor, a receptorfragment, a receptor subunit, a receptor subunit fragment, a ligand, aninhibitor, a hormone, a binding site, a lectin, a polyhistidine, acoupling domain, an oligonucleotide, a viral protein or a combination ofany two or more thereof.

Specifically, the capture element can be a SARS-CoV-2 viral structuralprotein. SARS-CoV-2 structural proteins include nucleocapsid protein(NP), membrane protein (MP), spike protein (SP), or epitopes thereof.The capture element may be NP/MP/SP or a fragment of NP/MP/SP.

As used herein, the terms “biomarker” refers to any substance used as anindicator of a biologic state. Thus, a biomarker can be any substancewhose detection indicates a particular disease state (for example, thepresence of an antibody may indicate an infection). Furthermore, abiomarker can be indicative of a change in expression or state of aprotein that correlates with the risk or progression of a disease, orwith the susceptibility of the disease to a given treatment. Once aproposed biomarker has been validated, it can be used to diagnosedisease risk, presence of disease in an individual, or to tailortreatments for the disease in an individual (e.g., choices of drugtreatment or administration regimes). In evaluating potential drugtherapies, a biomarker may be used as a surrogate for a natural endpointsuch as survival or irreversible morbidity. If a treatment alters thebiomarker, which has a direct connection to improved health, thebiomarker serves as a “surrogate endpoint” for evaluating clinicalbenefit. In one aspect, the target analyte is a biomarker.

In one embodiment, the target analyte is selected from a protein, aprotein fragment, a peptide, a polypeptide, a polypeptide fragment, anantibody, an antibody fragment, an antibody binding domain, an antigen,an antigen fragment, an antigenic determinant, an epitope, a hapten, animmunogen, an immunogen fragment, a virus protein, a virus coat protein,a virus, a virus protein or epitope thereof or any combination of anytwo or more thereof.

In one aspect, the target analyte is a SARS-CoV-2 antibody, antibodyfragment or binding domain thereof.

Capture elements specific for a target analyte are used to detect thepresence or absence of the analyte in a sample. A wide range ofcomplementary binding or coupling partners are known, with the choice ofcapture elements determined by the analytes to be detected, therequirement for adapter molecules and the level of specificity requiredfor the assay. In various aspects, the capture elements are specific forbinding/detecting IgG or IgM antibodies produced by a SARS-CoV-2infection.

The term “control element” refers to an element that is used to provideinformation on the function of the assay, for example bindingspecificity, the level of non-specific background binding, the degree ofbinding cross-reactivity, and the performance of assay reagents and thedetection system. Preferred controls useful herein include at least onenegative control to monitor background signal, at least one negativecontrol to monitor assay specificity, at least one positive colorimetriccontrol, and at least one positive control to monitor assay performance.

The substrate of the invention comprises at least one fiduciary markerthat will always be detectable on the substrate, preferably detectableirrespective of the performance of the assay or processing of thesubstrate.

The term “fiduciary marker” refers to a colored marker or label thatwill always be detectable on the substrate, preferably irrespective ofthe performance of the assay or processing of the substrate. The use ofat least one fiduciary marker will obviate the necessity of this elementbeing detected based on successful array processing, in comparison tothe positive colorimetric controls. The fiduciary marker is therefore a“true” positive control that would always be detectable regardless ofarray processing, and can be used to orient and help to grid the array.

In preferred aspects, the fiduciary marker is a dye, dye-conjugatedprotein or a chromogenic protein such as hemoglobin.

The term “negative control” refers to an element comprising print bufferor an unrelated protein to which no complementary binding partner isintended to be present in the assay. Any detectable signal from thenegative control can be used to determine the background threshold ofthe assay and the accuracy of any positive results. In one aspect, thenegative control to monitor background signal is print buffer. The printbuffer is a solution used to carry and print the capture elements andcontrol elements onto the substrate and may comprise buffered saline,glycerol and a surfactant, preferably a polysorbate surfactant such asTween 20. The blocking solution is used to reduce non-specific proteinbinding to the substrate surface and preferably comprises skim milk,casein, bovine serum albumin, gelatins from fish, pigs or other species,dextran or any mixture of any two or more thereof, preferably in asolution of phosphate buffered saline and a surfactant such as Tween 20.

The term “control capture element” refers to a capture element thatfunctions as a control, either a negative control that should not bindany analyte or a positive control that will bind a non-target analyte.

The substrate of the invention also comprises at least one control tomonitor assay performance. The control is intended to provideinformation of the efficiency of the complementary binding interactionsor the quality or performance of the reagents used.

The term “control to monitor assay performance” refers to an elementthat forms one part of a complementary binding interaction during anassay and is intended to provide information on the accuracy of theassay result. In one embodiment, the positive control to monitor assayperformance comprises one binding partner of a complementary bindingpair, where the other binding partner is a sample component or an assayreagent. The assay performance control is preferably selected from atarget analyte, a binding partner corresponding to and able to bind anon-target analyte that will be present in the sample, a binding partnercorresponding to and able to bind an assay reagent, and a colorimetricenzyme label, or any combination of any two or more thereof. An exampleof a binding partner corresponding to and able to bind a non-targetanalyte that will be present in the sample is an anti-Ig antibody thatwill bind an immunoglobulin present in a serum sample, thereforeconfirming a sample has been added. An example of a binding partnercorresponding to and able to bind an assay reagent is an anti-Igantibody that will bind a secondary immunoglobulin that is used toprocess the assay, such as biotinylated anti-target analyte antibody.Another example of a binding partner corresponding to and able to bindan assay reagent is a biotinylated antibody that will bind astreptavidin-peroxidase conjugate that is used to process the assay.

In one aspect, the assay performance control comprises one bindingpartner of a complementary binding pair, wherein the other bindingpartner is an assay reagent. The assay performance control is preferablyselected from the list comprising the target analyte, a non-specificbinding partner or a colorimetric enzyme label.

In another aspect, the complementary binding partners compriseantibody-antigen interactions or antibody-ligand interactions.

The substrate of the invention also comprises at least one control tomonitor assay specificity. The control is intended to provideinformation of the specificity of binding between the capture elementand the target analyte, or between the binding partners of the assaydetection steps.

The term “control to monitor assay specificity” refers to an elementthat is closely related to at least one binding partner of acomplementary binding pair present in the assay and is intended toprovide information of the specificity of the complementary binding.This control is a negative control that is not expected to generate adetectable result during normal assay processing. For example, in anantigen array for antibody detection, the assay specificity controlwould comprise an antigen that should not bind any antibody in thesample.

In one aspect, the assay specificity control comprises one or moreantibody isotypes, a corresponding antibody or antibody isotype from adifferent animal species or a closely related ligand. For example, inhuman antibody arrays, human IgM and anti-human IgM can be used ascontrols to monitor assay specificity.

The term “positive colorimetric control” as used herein refers to anenzyme or enzyme conjugate that provides a detectable signal uponaddition of the enzyme substrate.

In one embodiment, the positive colorimetric control is an enzyme labelconjugate capable of reacting with a colorimetric substrate, comprisingan enzyme selected from the list comprising horseradish peroxidase,alkaline phosphatases, β-D-galactosidase or glucose oxidase.

The identity of the assay controls will be dependent on the type ofarray, the identity of the target analyte, and the type of sample to beanalyzed.

For example, either anti-human IgG-HRP or anti-mouse IgG-HRP may be usedin arrays printed with antigens and antibodies, respectively. The finaldetection antibody in antigen arrays will often be anti-human IgG-HRP,while for antibody arrays it will often be a biotinylated mouse IgG.These controls can provide a positive control in addition to providinginformation on the performance or quality of the HRP substrate.

Mouse IgG, human IgG and anti-human IgG present on antigen or antibodyarrays can act either as positive or negative controls depending on thearray format, in addition to providing information of assay specificity.For example, mouse IgG should provide the positive signal in antibodyarrays, while the latter two should provide a positive signal in antigenarrays. These controls can also serve as controls for overall assayperformance.

The terms “sample” and “specimen” as used herein are used in theirbroadest sense to include any composition that is obtained and/orderived from biological or environmental source, as well as samplingdevices (e.g., swabs) which are brought into contact with biological orenvironmental samples. “Biological samples” include body fluids such asurine, blood, plasma, fecal matter, cerebrospinal fluid (CSF), andsaliva. In one embodiment, the biological sample is a cell, tissue, andor fluid obtained from a mammal, including from the upper respiratorytissues (such as nasopharyngeal wash, nasopharyngeal aspirate,nasopharyngeal swab, and oropharyngeal swab), from the lower respiratorytissues (such as bronchiolar lavage, tracheal aspirate, pleural tap,sputum), blood, plasma, serum, and stool. These examples areillustrative, and are not to be construed as limiting the sample typesapplicable to the present invention.

In various aspects of the present invention, the sample is a bloodsample, including a plasma or serum sample.

The assay techniques used in conjunction with the substrates of thepresent invention include any of a number of well-known colorimetricenzyme-linked assays. Examples of such systems are well known in theart. The assay techniques are based upon the formation of a complexbetween a complementary binding pair, followed by detection with acolorimetric detection system comprising an enzyme-conjugate label and acolorimetric substrate. The detection system will be described withreference to enzyme-linked immunosorbent assays (ELISA), though askilled person would appreciate that such techniques are not restrictedto the use of antibodies but are equally applicable to any colorimetricassay.

In one embodiment, the ELISA is in the “sandwich” assay format. In thisformat, the target analyte to be measured is bound between twoantibodies—the capture antibody and the detection antibody. In anotherembodiment, the ELISA is a non-competitive assay, in which an antibodybinds to the capture antigen and the amount of bound antibody isdetermined by a secondary detection antibody.

Either monoclonal or polyclonal antibodies may be used as the captureand detection antibodies in sandwich ELISA systems. Monoclonalantibodies have an inherent monospecificity toward a single epitope thatallows fine detection and quantitation of small differences in antigen.A polyclonal antibody can also be used as the capture antibody to bindas much of the antigen as possible, followed by the use of a monoclonalantibody as the detecting antibody in the sandwich assay to provideimproved specificity. A monoclonal antibody can also be used as thecapture antibody to provide specific analyte capture, followed by theuse of a polyclonal antibody as the detecting antibody in the sandwichassay. Additionally, both the capture and the detection antibodies couldbe monoclonal.

The term “antibody” as used herein includes naturally occurringantibodies as well as non-naturally occurring antibodies, including, forexample, single chain antibodies, chimeric, bifunctional and humanizedantibodies, as well as antigen-binding fragments thereof. Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly or can be obtained, forexample, by screening combinatorial libraries consisting of variableheavy chains and variable light chains (see Huse et al., Science246:1275-1281, 1989, which is incorporated herein by reference). Theseand other methods of making, for example, chimeric, humanized,CDR-grafted, single chain, and bifunctional antibodies are well known(Winter and Harris, Immunol. Today 14:243-246, 1993; Ward et al., Nature341:544-546, 1989; Harlow and Lane, Antibodies: A laboratory manual(Cold Spring Harbor Laboratory Press, 1999); Hilyard et al., ProteinEngineering: A practical approach (IRL Press 1992); Borrabeck, AntibodyEngineering, 2d ed. (Oxford University Press 1995); each of which isincorporated herein by reference). In addition, modified or derivatizedantibodies, or antigen binding fragments of antibodies, such aspegylated (polyethylene glycol modified) antibodies, can be useful forthe present methods. As such, Fab, F(ab′)2, Fd and Fv fragments of anantibody that retain specific binding activity are included within thedefinition of an antibody.

The term “secondary antibody” refers to an antibody that will bind atarget analyte and that is conjugated with either an adaptor moleculesuch as biotin or an enzyme label such as horseradish peroxidase (HRP).Antibody-adaptor conjugates are processed to give a detectable result bycontacting the antibody-adaptor conjugate with an adaptor-enzymeconjugate and then the enzyme substrate; for example, antibody-biotinconjugates will bind streptavidin-HRP conjugates. Antibody-enzyme labelconjugates include antibody-HRP conjugates. Use of secondary antibodiesis discussed and exemplified below.

The term “binds specifically” or “specific binding activity” or thelike, means that two molecules form a complex that is relatively stableunder physiologic conditions. The term is also applicable where, anantigen-binding domain is specific for a particular epitope, which iscarried by a number of antigens, in which case the antibody carrying theantigen-binding domain will be able to bind to the various antigenscarrying the epitope. Specific binding is characterized by a highaffinity and a low to moderate capacity. Typically, the binding isconsidered specific when the affinity constant is about 1×10⁻⁶ M,generally at least about 1×10⁻⁷ M, usually at least about 1×10⁻⁸M, andparticularly at least about 1×10⁻⁹ M or 1×10⁻¹⁰ M or less.

After array manufacture and prior to sample addition, all availableprotein-binding sites on the substrate surface are blocked by additionand incubation with one or a combination of reagents. These reagents arecalled “Blockers” and serve to decrease or at best eliminatenon-specific protein binding from the sample on the substrate surfacethereby decreasing overall background signal. This increases the ratioof signal to noise, thereby increasing the overall sensitivity of theassay. Blockers play no active part in the subsequent reactions betweenthe sample and other assay reagents and the immobilized proteins on thesubstrate. Exemplary blockers include, but are not limited to, bovineserum albumin, casein, non-fat dry milk, gelatin derived from fish, pigsand other sources, dextran, serum derived from sources other than thesample being analyzed such as from steelhead salmon, guinea pigs,hamsters, rabbit and other sources, polyethylene glycol, polyvinylpyrrollidone, and commercial preparations including HeteroBlock (OmegaBiologicals, Bozeman, Mont.), SuperBlock, StartingBlock, SEA BLOCK(Pierce, Rockford, Ill.). Typically, blockers are made up in buffersolutions such as, for example, phosphate buffer, phosphate bufferedsaline, Tris buffer, acetate buffer and others. The blockers may also besupplemented with detergents such as, for example, Tween 20, Tween 80,Nonidet P40, sodium dodecyl sulfate and others.

An important consideration in designing an array is that the capture anddetection antibodies of each binding pair must recognize twonon-overlapping epitopes so that when the antigen binds to the captureantibody, the epitope recognized by the detection antibody must not beobscured or altered. A large number of complementary binding pairs havealready been developed for ELISA and can be used in the presentinvention.

For multiplexed assays, it is also important that there is no overlapbetween each of the binding pairs to eliminate cross-reactivity. Anumber of multiplexed ELISAs have been developed and it is anticipatedother combinations of binding pairs could be configured through testing.

In one aspect, the enzyme-conjugate label comprising an enzyme selectedfrom the list comprising horseradish peroxidase, alkaline phosphatase,β-D-galactosidase or glucose oxidase.

In an additional aspect, the enzyme label may be conjugated directly toa primary antibody or introduced through a secondary antibody thatrecognizes the primary antibody. It may also be conjugated to a proteinsuch as streptavidin if the primary antibody is biotin labelled.

In a further aspect, the assay detection system comprises a detectioncolorimetric substrate selected from the list comprising 3,3′,5,5′-tetramethylbenzidine, diaminobenzidine, metal-enhanceddiaminobenzidine, 4-chloro-1-naphthol, colloidal gold, nitro-bluetetrazolium chloride, 5-bromo-4-chloro-3′-indolylphosphate p-toluidinesalt and naphthol AS-MX phosphate+Fast Red TR Salt.

In certain aspects, the colorimetric reaction can be detected andoptionally quantified and analyzed using an image capture device such asa digital camera or a desktop scanner attached to a computer. Knownmethods for image analysis may be used. For example, the concentrationvalues of known standard elements can be used to generate standardcurves. Concentration values for unknown analytes can be analyzed usingthe standard curve for each analyte to calculate actual concentrations.Values for each analyte can be identified based on the spotting positionof each capture element within the array.

The substrates of the present invention are particularly amenable to usein kits for the detection of target analytes. Such kits may comprise thesubstrates together with instructions and any assay consumablesrequired. Different kits are envisaged for different target analytes andtypes of array. Accordingly, in an additional embodiment, the presentinvention provides a kit for detecting a plurality of target analytes ina sample, containing a substrate and optionally one or both of abackground reducing reagent, and a colorimetric detection system. In oneaspect, the kit also contains one or more items from a wash solution,one or more antibodies for detection of antigens, ligands or antibodiesbound to the capture elements or for detection of the positive controls,software for analyzing captured target analytes, and a protocol formeasuring the presence of target analytes in samples. In an additionalaspect, the antibodies for detection are antibody-binding protein (BP)conjugates, antibody-enzyme label conjugates, or any combinationthereof. In a further aspect, the sample is a blood sample, e.g., serumor plasma. In one aspect, the substrate is a solid or a poroussubstrate. In an additional aspect, the solid substrate is aparamagnetic bead, microtiter plate, microparticle, or a magnetic bead.In certain aspects, the porous substrate is a membrane.

In another aspect, the invention also relates to a method of processinga substrate of the invention. Such a method comprises providing asubstrate of the invention as described above, adding at least onesample to the substrate, and processing the substrate such that adetectable result is given by two or more of at least one fiduciarymarker, at least one positive colorimetric control, and iii) at leastone positive control to monitor assay performance.

In another aspect, the present invention provides methods for processinga microarray by providing a substrate, adding at least one sample to thesubstrate, and processing the substrate such that a detectable result isgiven by two or more of at least one fiduciary marker, at least onepositive colorimetric control, and at least one positive control tomonitor assay performance.

In one aspect, the step of processing the substrate or microarraycomprises a blocking step during which available protein-binding siteson the substrate or microarray are blocked with a blocker, an optionalwash step, contacting the substrate or microarray with the samplecontaining the one or more analytes to be measured, a wash step toremove non-bound material from the substrate or microarray, contactingthe substrate or microarray with one or more secondary antibodies thatcorrespond to and will bind one or more target analytes and non-targetanalyte that is bound to an assay performance control, a wash step, andcontacting the substrate or microarray with one or both of an enzymeconjugate or an enzyme substrate to generate a detectable result.

In one embodiment, the present invention provides methods for detectingan analyte in a sample comprising providing a substrate, adding at leastone sample to the substrate, and processing the substrate such that adetectable result is provided. In one aspect, the detectable resultincludes two or more of at least one fiduciary marker, at least onepositive colorimetric control, and at least one positive control todetect an analyte in the sample.

In another aspect, the substrate of the invention can be used for thesimultaneous detection of at least one target analyte in a sample, andpreferably a plurality of different target analytes in a sample, andhave utility in diagnostic and screening assays.

Thus, the substrates of the invention provide the advantage that theycan be adapted to high throughput (or ultra high throughput) analysisand, therefore, any number of samples (e.g., 96, 1024, 10,000, 100,000,or more) can be examined in parallel, depending on the particularsupport used. A particular advantage of adapting the substrates to highthroughput analysis is that an automated system can be used for addingor removing reagents from one or more of the samples at various times,for adding different reagents to particular samples, or for subjectingthe samples to various heating cycles.

For example, the automated system may consist of one or moretemperature-controlled chambers and one or more robotic arms mounted ona deck that has platforms configured to hold 96-well plates. Themovement of the robotic arms and the temperature in the chambers arecontrolled by a central computer unit. The array plates are stacked onthe deck of the instrument. In one embodiment, the plates containingsamples to be analyzed are stacked in a chamber with temperature of 4°C. One robotic arm then sequentially transfers each individual arrayplate on one platform while the other arm sequentially transfers eachindividual sample plate on the second platform. A nozzle containing 96disposable tips then aspirates a predetermined volume of sample fromeach well of the sample plate and transfers the sample to thecorresponding wells of the array plate. The array plate containing thesample is then transferred to a chamber with temperature of 37° C. Thisprocess is repeated until sample has been added to all the array platesstacked on the deck. The array plates are incubated for a predeterminedtime followed by transfer of each plate to the platform for addition ofwash buffer with the nozzle containing 96 disposable tips. The washbuffer is aspirated after a predetermined time and this wash process isrepeated multiple (i.e., two or more) times. Each array plate thenreceives the secondary antibody followed by transfer to a chamber withtemperature of 37° C. The array plates are incubated for a predeterminedtime followed by transfer of each plate to the platform for addition ofwash buffer with the nozzle containing 96 disposable tips. The washbuffer is aspirated after a predetermined time and this wash process isrepeated multiple (i.e., two or more) times. Each array plate thenreceives the detection reagent followed by incubation for apredetermined time followed by transfer of each plate to the platformfor addition of wash buffer with the nozzle containing 96 disposabletips. The wash buffer is aspirated after a predetermined time and theplate transferred to the 37° C. chamber for drying. The plates aretransferred back to the deck after a predetermined period and manuallyprocessed for analyses of data.

In addition to the convenience of examining multiple test agents and/orsamples at the same time, such high throughput assays provide a meansfor examining duplicate, triplicate, or more aliquots of a singlesample, thus increasing the validity of the results obtained, and forexamining control samples under the same conditions as the test samples,thus providing an internal standard for comparing results from differentassays.

In a further embodiment, the present invention provides methods ofdetecting exposure of a subject to SARS-CoV-2 by contacting a substratewith a biological sample from the subject, wherein the subject issuspected of having COVID-19; and detecting the presence of an antibodythat binds to SARS-CoV-2,. In one aspect, the detection method isautomated, manual, lateral flow, solid-phase, chemiluminescence,microfluidics, lab-on-a-chip based immunoassay, ELISA, or a combinationthereof. In an additional aspect, the antibody is IgG and/or IgM. In afurther aspect, the sample is a blood sample, for example serum orplasma.

The present invention provides methods of in vitro diagnosticapplications for the detection of antibodies produced in response toSARS-CoV-2 infection such as manual multiplex immune assay, automatedmultiplex immune assay (MIA), manual singleplex ELISA (solid phase),automated chemiluminescent immune assay (CLIA), wash-free immune assays(manual and automated), automated centrifugal microfluidics-basedimmunoassay, lab-on-a-chip based immunoassay, paramagnetic bead-basedmanual ELISA, manual paper-based ELISA, Point-of-care (POC) immunoassaysand other immunoassay formats. In one aspect, detection is bycolorimetric imaging (e.g., PictArray™, U.S. Pat. No. 9,625,453),absorbance (e.g., manual ELISA), chemiluminescence (e.g., automatedCLIA, CRET), florescence (e.g., manual immunoassays, ELISA, FRET)and bythe naked eye (e.g., lateral flow immunoassays).

The COVID-19 MIA can be used for the clinical diagnosis of SARS-CoV-2infected persons using different immunoassay formats. For example, theCOVID-19 MIA can be performed using a membrane (e.g., PictArray™, U.S.Pat. No. 9,625,453) or on the solid surface of 96-well microtiter plate(MTP). The antibodies (IgG and IgM) in the SARS-CoV-2 infectedindividuals can be detected via an indirect immunoassay, where multiplerecombinant structural proteins of SARS-CoV-2, e.g., NP, SP and/or MP,will be coated on the membrane or the solid surface of MTP. The COVID-19MIA can be automated using an analyzer that automates all the steps inthe manual MIA and uses a colorimetric reader and image analysissoftware (e.g., PictImager™ and Pictorial©). The COVID-19 MIA can beperformed as a lateral flow immunoassay (LFIA) or a manual singleplexELISA assay.

Further, the COVID-19 MIA can be performed using chemiluminescentimmunoassays (CLIAs), both multiplex as well as singleplex. The multiplestructural proteins of SARS-CoV-2 can be bound covalently toparamagnetic beads (micron-sub-micron size) and used for the detectionof IgG and IgM antibodies against SARS-CoV-2 via indirect immunoassay.The detection signal is generated by conjugating the detection antibodywith acridinium or other chemiluminescent labels and generating achemiluminescent signal.

The COVID-19 MIA can be performed by manual and automated wash-freeassays for the detection of IgG and IgM.

The COIVD-19 MIA can also be performed as a wash-freeelectrochemiluminescent ELISA. The analytes in sample can be detectedusing biomolecule-coated (antigen-coated) carbon electrode surface-basedmicrowell plates and SULFO-TAG-labelled detection Ab that emits lightupon electrochemical stimulation.

Further, the COVID-19 MIA can be performed by centrifugalmicrofluidics-based automated immunoassay. The multiple structuralproteins of SARS-CoV-2 can be covalently bound to paramagnetic beads andused for the detection of IgG and IgM antibodies generated in thesubjects in response to SARS-CoV-2 infection. The detection of analyteoccurs in a reaction chamber, followed by washing the specific immunecomplexes formed on paramagnetic beads and transfer of the paramagneticbeads to the detection chamber for the generation and reading of assaysignal, which can be chemiluminescence, absorbance or fluorescence.

The COVID-19 MIA can also be performed as a lab-on-a-chip (LOC) assay.Paramagnetic beads or solid surfaces can be used for the covalentattachment of multiplex structural proteins of SARS-CoV-2. The detectionsignal could be chemiluminescent, fluorescent, absorbance,electrochemical or colorimetric

Additionally, the COVID-19 MIA can be performed using manual singleplexELISA. Paramagnetic beads can be bound covalently to multiplexstructural proteins of SARS-CoV-2 to detect antibodies raised againstSARS-CoV-2.

Further, the COVID-19 MIA can be a point of care (POC) immunoassay. ThePOC immunoassay can be label-free using a disposable strip, where theantibodies are detected using electrochemical reader or asmartphone-based reader.

In additional to the assay formats mentioned above, the COVID-19 MIA canbe performed using wash-free immunoassay based on fluorescence resonanceenergy transfer (FRET) or chemiluminescence resonance energy transfer(CRET); signal-enhanced immunoassay based on the use ofnanoparticle-based signal detection step or the use of micro- andsubmicro-beads for binding capture antibodies/antigens; and rapidmultiplex immunoassays based on Lab-in-a-tube technology or verticalmicrofluidics.

The following examples are provided to further illustrate theembodiments of the present invention, but are not intended to limit thescope of the invention. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used

EXAMPLES Example 1 COVID-19 MIA

The COVID-19 MIA format involves the detection of IgG and/or IgMantibodies (Ab) generated in humans after exposure to the SARS-CoV-2virus.

The format will spot the SARS-CoV-2 structural proteins (e.g.,Nucelocaspid protein (NP), spike protein (SP) and membrane protein (MP))onto either membrane (e.g., nitrocellulose, nylon; such as PictArray™,U.S. Pat. No. 9,625,453) or membrane-free (e.g., polystyrene microtiterplate) assay surfaces. The capture antigens (Ag) and Ab as well as thedetection Ab, used for the development of the COVID-19 MIA have alreadybeen identified, as shown in Table 1.

TABLE 1 Assay materials identified to be screened for use for theCOVID-19 MIA Table of assay materials used Vendor Cat # Product Name 1.Nucleocapsid Protein antigen Bio Bench nCoV-P003/XG01 NucleocapsidProtein, Fragment (N - protein) 2. Spike Protein antigen The NativeAntigen Company REC31806 SARS-CoV-2 Spike Glycoprotein (S1), SheepFc-Tag (HEK293) 3. Anti-Human detection antibody ImmunobioscienceSA-9001-12 Goat anti-Human IgG-HRP

The generalized assay format for the COVID-19 MIA is summarised inFIG. 1. The IgG and/or IgM antibodies against SARS-CoV-2 would bedetected by indirect IA (FIG. 1A) (FIG. 1B). The SARS-CoV-2 structuralproteins (NP, SP and MP) will be printed as spots onto the assay surfaceusing a microarray printer in a single well. Additionally, NP, SP and MPcan either be printed as separate discrete spots or a mixture of allthree viral structural proteins printed as a single spot.

The COVID-19 kit will involve the printing of SARS-CoV-2 structuralproteins on the assay surface (FIG. 2A) followed by the blocking of eachwell with an appropriate blocking solution to obviate any non-specificbinding (FIG. 2B). The developed printed array will be supplied to theend-users along with the assay components for the detection Ab againstSARS-CoV-2. Internal positive and negative controls will also besupplied which can be tested alongside patient samples to ensure optimalassay performance. Immunoassay (IA) procedures for the detection of Abwill involve the addition of diluted patient serum to the well(s) andincubating at 37° C. for tens of minutes so that specific immunecomplexes are formed between the Ag and Ab, e.g., binding of IgG/IgM toSARS-CoV-2 structural proteins (FIG. 2C). The excess andnon-specifically bound analytes are then taken away by washing the wellswith wash buffer. Subsequently, HRP-labelled detection Ab, e.g.,anti-human IgG/IgM HRP and anti-NP HRP are added to the wells andincubated at 37° C. for tens of minutes (FIG. 2D). It results in theformation of biomolecular immune complexes between detection Ab andCOVID-19 analytes. This is followed by second washing step with washbuffer that removes the excess and non-specifically bound analytes fromthe wells. Finally, HRP substrate is added to the wells and incubatedfor some minutes at room temperature. It leads to the formation ofcolorimetric array spots via the precipitation of the colorimetricproduct produced after the enzyme substrate reaction if the targetanalytes are present in the patient serum (FIG. 2E). This is followed bythird washing step with wash buffer. In case of membrane-based IA,3,3′-Diaminobenzidine (DAB) will be used as the HRP substrate and afterincubation, the wells will be washed with wash buffer and then dried at37° C. before analysis. But for membrane-free IA, a precipitating3,3′,5,5′-Tetramethylbenzidine (TMB) solution will be used as the HRPsubstrate and after incubation, the wells will be washed once prior toanalysis.

The COVID-19 MIA results in the formation of colorimetric spots, theintensity of which is directly proportional to the concentration of Ab(IgG and/or IgM) present in the patient sample. Depending on the assaysurface used, the colorimetric arrays are imaged by using indigenouslydeveloped handheld or portable benchtop colorimetric reader device. Itcan also be read by the commercial colorimetric readers, such as thoseavailable from Scienion AG, Germany.

Imaging software analyses the images from the multiplex colorimetricreaders to detect the microarray spots from each well and generates theresults as output. The software first identifies the wells and thendetects the positive control spots within each well that appears afterthe MIA. The positive control spots act as alignment anchors, which areused by the software to place a microarray grid for all spots withineach well. The software algorithm then analyses the colorimetric imageof the array and extracts the pixel intensity for each spot. The datagenerated from each spot is then collated with the layout of array andpatient samples to provide a final test report for the samples beinganalysed. An example of such imaging software is Pictorial©

Example 2 COVID-19 MIA—Membrane-Based, Single Well

The COVID-19 MIA—Membrane-based, Single well format utilises the 16-wellarray slide containing membrane disks affixed to the bottom of each wellas the assay surface. SARS-CoV-2 structural proteins (NP, SP and MP) areall immobilised in duplicate as separate spots in each well.

The patient samples are diluted and added to the array slide andincubated at 37° C. then washed with wash buffer. HRP labelledanti-Human IgG and/or IgM detection Ab is then added to all wells of thearray slide. The wells are incubated at 37° C. and then washed followedby the addition of DAB substrate. After a short incubation, the wellsare washed once and then dried at 37° C. prior to analysis.

Example 3 COVID-19 MIA—Membrane-Based, Single Well, Using GroupedSARS-CoV-2 Structural Proteins

The COVID-19 MIA—Membrane-based, Single well, using grouped SARS-CoV-2structural proteins format utilises the 16-well array slide containingmembrane disks affixed to the bottom of each well as the assay surface.A mixture of SARS-CoV-2 structural proteins (NP, SP and MP) are allimmobilised in duplicate spots in each well.

The patient samples are diluted and added to the array slide, incubatedat 37° C., and then washed with wash buffer. HRP labelled anti-Human IgGand/or IgM detection Ab is then added to all wells of the array slide.The wells are incubated at 37° C. and then washed followed by theaddition of DAB substrate. After a short incubation, the wells arewashed once and then dried at 37° C. prior to analysis.

Example 4 COVID-19 MIA—Membrane-Free, Single Well

The COVID-19 MIA (Membrane-free, Single well) format utilises the96-well microtiter plate as the assay surface. For each well in the96-well plate, SARS-CoV-2 structural proteins (NP, SP and MP) areimmobilised in duplicate as separate spots.

The patient samples are diluted and added to the 96-well plate,incubated at 37° C., and then washed with wash buffer. HRP labelledanti-Human IgG and/or IgM detection Ab is then added to all wells. Thewells are incubated at 37° C. and then washed followed by the additionof TMB substrate. After a short incubation, the wells are washed onceand then analysed.

Example 5 COVID-19 MIA—Membrane Free, Single Well, Using GroupedSARS-CoV-2 Structural Proteins

The COVID-19 MIA (Membrane-free, Single well, using grouped SARS-CoV-2structural proteins) format utilises the 96-well microtiter plate as theassay surface. For each well in the 96-well plate, a mixture ofSARS-CoV-2 structural proteins (NP, SP and MP) are immobilised induplicate.

The patient samples are diluted and added to the 96-well plate,incubated at 37° C., and then washed with wash buffer. A mixture of HRPlabelled anti-NP detection Ab and HRP labelled anti-Human IgG and/or IgMdetection Ab is added to all wells. The wells are incubated at 37° C.and then washed followed by the addition of TMB substrate. After a shortincubation, the wells are washed once and then analysed.

Example 6 Automated COVID-19 MIA—Membrane Free, 96-Well MTP

The automated MIA will be performed inside an analyzer, where all thesteps in the manual MIA will be automated. The dispensing and aspirationof reagents is done by a needle attached to the robotic arm. The assaycomponents will be provided in the form of ready-to-use assay cartridgesthat are simply plugged inside the analyzer and can perform up to 100MIA tests. The washing of the MTP wells will be done by the roboticneedle using specific washing programs. Similarly, the needle will bewashed after each dispensing step. However, disposable tips could alsobe used, which would obviate the cleaning of the needle after eachdispensing step. All the steps of the IA will be optimized for theautomated MIA. The readout of the colorimetric array spots in theprocessed 96-well MTP will be performed using an integrated colorimetricreader and an image analysis software. The analyzer would have adedicated compartment for putting the patient sample vials, anddedicated spaces for putting the wash buffer, TMB substrate and otherbuffers. The analyzer would need to undergo daily, weekly and monthlymaintenance.

Example 7 Automated Chemiluminescent Immunoassay (CLIA)

The assay formats used for development of MIA could be further employedfor the development of automated CLIAs, both multiplex as well assingleplex, for the diagnosis of SARS-CoV-2 infection. The multiplestructural proteins of SARS-CoV-2 could be bound covalently toparamagnetic beads (micron-sub-micron size) and used for the detectionof IgG and IgM antibodies against SARS-CoV-2 via indirect immunoassay.The magnetic beads could be provided with a mixture of multiplestructural proteins or various formulations of magnetic beads could becoated with each of the structural proteins and then mixed together forthe assays. It could be a total IgG+IgM antibody test or IgG and IgMcould be detected separately. The detection signal in case of automatedCLIAs could be generated by conjugating the detection antibody withacridinium or other chemiluminescent labels and providing theappropriate trigger solutions for the generation of chemiluminescentsignal. All the automated CLIAs are performed using a high-throughputanalyzer. The assay reagents are stored in the form of assay cartridgesthat can used for up to 100 tests. The buffers, wash solution andtrigger solutions are stored at the respective places in the analyzer.The patient sample vials are placed inside the analyzer at a dedicatedplace while the assay/reaction vials are provided automatically asconsumable for each CLIA test.

Example 8 COVID-19 ELISA

Manual ELISA can be developed for the detection of IgM and IgG using thedeveloped MIA procedure with customization of some steps for ELISA. The96-well MTP would be coated with a mixture of NP, SP and/or MP eitherpassively or using a leach-proof biomolecular immobilization procedurebased on silane chemistry (Vashist et al., Sci Rep, 4:4407, 2014, DOI:10.1038/srep04407). All the immunoassay steps for the detection of IgGand IgM ELISAs would then be performed exactly as specified in the MIAexcept the last step. In case of ELISA, the signal would be generated byenzyme-substrate reaction by providing TMB and H₂O₂ to the HRP-labeleddetection Ab. The enzyme-substrate reaction is stopped by providing astop solution comprising of 1N H₂SO₄. The optical density of thecolorimetric solution is then read at 450 nm with reference at 650 nm.The detection of IgG and IgM is done by indirect assay

Materials. Reagents for the detection of IgG: SARS-CoV-2 NP, SARS-CoV-2SP and, goat anti-human IgG-HRP. NP; and HRP labeled rabbit/mouseanti-SARS CoV Ab (detection Ab).

Reagent set up: PBS: Add a BupH phosphate buffered saline pack to 100 mLof autoclaved DIW, dissolve well and make the volume up to 500 mL usingautoclaved DIW. Each pack makes 500 mL of PBS at pH 7.2, which can bestored at room temperature (RT) for a week and at 4° C. for up to fourweeks. APTES: The procured APTES solution has a purity of 99%.Reconstitute in autoclaved DIW to make 1% (v/v) APTES just before mixingwith capture anti-HFA Ab.

Example 9 COVID-19 IgG/IgM ELISA

Mix COVID-19 structural antigens solution (mixture of NP, SP and/or MP)with 0.5-2% (v/v) APTES in the ratio of 1:1 (v/v). Incubate each of thedesired wells of a 96-well MTP with 100 μL of the freshly preparedanti-NP capture Ab solution for 30 min at RT. Wash five times with 300μL of 0.1M PBS, pH 7.4. Washing can also be performed with an automaticplate washer. (Passive Ab immobilization, by incubating with the Abovernight at 4° C., could also done). Block the COVID-19 Ag-bound wellswith 300 μL of 1-5% (w/v) BSA for 30 min at 37° C. followed by extensivePBS washing (as mentioned previously). Add 100 μL of varying humanIgG/IgM concentrations or the patient serum/plasma sample (dilution tobe determined after optimization) to different BSA-blocked wells.Incubate for 1 h at 37° C. and wash extensively with PBS (as statedpreviously). Add 100 μL of HRP-labeled anti-human IgG/IgM detection Ab(200 ng mL⁻¹) in each of the NP-captured wells. Incubate for 1 h at 37 °C. and wash extensively with PBS. Add 100 μL of TMB-H₂O₂ mixture to eachof these wells and incubate at RT to develop color for 15 min. Stop theenzyme-substrate reaction by adding 50 μL of 1 N H₂SO₄ to each well.Determine the absorbance at a primary wavelength of 450 nm taking 540 nmas the reference wavelength in a microplate reader.

Example 10 Rapid One Step Kinetics-Based ELISA

A customized rapid one step kinetics-based rapid ELISA procedure couldbe employed for the detection of IgG/IgM, as specified in Vashist etal., Biosensors and Bioelectronics 67, 73-78, 2015.

Example 16 Rapid One Step Kinetics-Based ELISA Using Paramagnetic Beads

A customized rapid one step kinetics-based ELISA procedure could bedeveloped using paramagnetic beads for the detection of IgG/IgM, asspecified in Vashist et al., Analytical Biochemistry 456, 32-37, 2014.

Example 17 Centrifugal Microfluidics-Based Automated Point-of-CareImmunoassy

A customized centrifugal microfluidics-based automated point-of-careimmunoassay procedure could be developed using paramagnetic beads forthe detection of IgG/IgM, as conceived in Czilwik et al., RSC Advances5(76), 61906-61912, 2015.

Example 18 Wash-Free Immunoassay

Manual and automated wash-free MIAs could be developed for the detectionof IgG and IgM. As an example, the IA for IgG/IgM would involve thespecific biomolecular interactions of NP/SP/MPcoated donor beads withanother goat/rabbit/mouse anti-human Ab-coated acceptor beads in thepresence of IgG/IgM in sample, which form immune complexes and generatea chemiluminescent signal as the donor and acceptor beads are inproximity. This format will substantially reduce the assay duration andcomplexity (no washing steps required) and would have high sensitivityand broad dynamic range.

Example 19 Summary of COVID-19 Multiplex Immunoassay (MIA)

The MIA format involves the simultaneous detection of IgG antibodiesgenerated in humans after exposure to the SARS-CoV-2 virus. The formatemploys PictArray™ technology for the spotting of SARS-CoV-2 structuralproteins (Nucleocapsid Protein (NP), spike protein (SP)) onmembrane-free (i.e., polystyrene) assay surfaces.

The overall assay format for the COVID-19 MIA is summarised in FIG. 1.The IgG antibodies against SARS-CoV-2 are detected via indirectimmunoassay. The SARS-CoV-2 structural proteins (NP and SP) are printedas spots on to the assay surface using a microarray printer, bothproteins are printed within the same assay well. Additionally, NP and SPcan either be printed as separate discrete spots or as a mixture of bothproteins printed as a single spot.

After printing SARS-CoV-2 proteins on the assay surface (FIG. 2A), eachwell will be blocked with an appropriate blocking solution to eliminateany non-specific binding (FIG. 2B). The assay is divided into threedistinct steps, with a duration of 1 hour and 5 minutes. In the firststep of the assay, diluted patient serum is added, and the wells areincubated at 37° C. for 30 minutes to allow the binding of targetanalytes, i.e., IgG antibodies to SARS-CoV-2 proteins (FIG. 2C). Thewells are washed with wash buffer to remove any unbound serumcomponents, followed by step two, HRP-labelled detection antibodies,i.e., anti-Human IgG-HRP, are added, and the wells are incubated at 37°C. for 15 minutes to allow the detection of the target analytes (FIG.2D). The wells are washed for a second time to remove any unbounddetection antibodies. In the third step, HRP substrate is added (aprecipitating 3,3′,5,5′-Tetramethylbenzidine (TMB) solution), and thewells are incubated at room temperature (21° C.-25° C). for 20 minutesto allow the visualisation of the array spots that the target analytespresent in the serum bind to (FIG. 2E).

Example 20 COVID-19 MIA—Membrane Based, SARS CoV-2, Structural ProteinsPrinted in Dublicate

The printed spots in the COVID-19 MIA are shown as shaded circles whereeach circle of a particular shade corresponds to a specific SARS-CoV-2structural protein that is printed on the membrane or the solid surface(FIG. 7A-B). The white circles signify that nothing has been printed atthat specific position.

The plate preparation and spotting were performed as follows. Eachantigen (SARS-CoV-2 NP and SARS-CoV-2 SP (S1)) was diluted in NP-400.05% (1 volume of NP-40 0.05% in 9 volumes of antigen) and incubatedfor 15 min at room temperature (20° C. to 25° C). , followed by theaddition of 2X print buffer (2X PB) and RO water. The final solution hasthe antigen concentrations at 200 μg/ml and 200 μg/ml in 1X print buffer(1X PB) for SARS-CoV-2 NP and SARS-CoV-2 SP, respectively. Biotinylatedgoat anti-mouse IgG is used as a positive control to confirm theaddition of the secondary antibody solution and also aids in thealignment of the image analysis software during the analysis of results.Biotinylated goat anti-mouse IgG is printed at a concentration of 20μg/ml, in PB and RO water. Print buffer is printed as the negativecontrol in the assay. It indicates the overall assay background. Twentyeight μL of each of the prepared proteins and print buffer solution weretransferred in a 384-well PCR plate for printing. The printing wasperformed using the Thomas™ microarrayer under the following conditions:temperature—23° C. and relative humidity—56%. The pin is washed in ROwater before each spot is printed. Visual quality control of the printedarray was performed to check spot positioning and morphology. The slideswere then dried for 30 minutes at 37° C. . Seventy five μL of blockingsolution was added to each slide well and incubated at 37° C. for 30minutes. Slides were inverted and tapped to remove the blocking solutionand dried for 15 minutes at 37° C. The dried slides were stored at 2-8°C. in a plastic box with desiccants.

Slides were brought to room temperature prior to be used. One volume ofserum/plasma sample was added to 9 volumes of assay diluent (1:10dilution). Fifty μL of diluted sample was dispensed in each well then,the slides were covered (using a plate cover) and incubated for 1 hourat 37° C., followed by washing 3 times using 604, of washing solutionper well for each wash. Any excess liquid was removed by inverting theslides. Fifty μL of the secondary antibody (0.5 μg/ml) was added to eachslide well, which was then cover and incubated for 30 minutes at 37° C.. The slide was washed using 604, of washing solution per well for threewash steps. Any excess liquid was removed by inverting the slide. FiftyμL of DAB substrate diluted in Hydrogen Peroxide (H₂O₂) (1 volume of DABin 19 volumes of H₂O₂) was added to each well of the slide and incubatedfor 5 minutes at room temperature in the dark. DAB was then removed byinverting and tapping the slide onto an absorbent tissue, the slideswere washed once using the washing solution, inverted and then dried 15minutes at 37° C. . The slides were read using the Pictlmager™ readerand the data automatically processed once the slides are scanned by thePictorial© software.

The COVID-19 MIA was assessed against the first WHO internationalreference panel for anti-SARS-CoV-2 immunoglobulin, which is made up offive referenced samples which have different reactivities against NP andSP, printed on our platform. Using SARS-CoV-2 Nucleocapsid Protein asthe target, the assay signals for the reference panel correlated to thereactivity levels indicated by the provider, i.e., the signal decreasedfrom the highly reactive sample to the low reactive one, and noreactivity for the negative sample (FIG. 8).

Example 21 COVID-19 MIA—Membrane Free, Single Well

The printed spots in the COVID-19 MIA are shown as shaded circles whereeach circle of a particular shade corresponds to a specific SARS-CoV-2structural protein that is printed on the membrane or the solid surface(FIG. 9A-B). The white circles signify that nothing has been printed atthat specific position.

The plate preparation and spotting were performed as follows. Eachantigen (SARS-CoV-2 NP and SARS-CoV-2 SP (S1)) was diluted in NP-400.05% (1 volume of NP-40 0.05% in 9 volumes of antigen) and incubatedfor 15 min at room temperature, followed by the addition of 2X printbuffer (2X PB) and RO water. The final solution has the antigenconcentrations at 100 μg/ml and 200 μg/ml in 1X print buffer (1X PB) forSARS-CoV-2 NP and SARS-CoV-2 SP, respectively. Biotinylated goatanti-mouse IgG, used as a positive control to confirm the addition ofthe secondary antibody solution, also aids in the alignment of the imageanalysis software during final result analysis. Biotinylated goatanti-mouse IgG is printed as the positive control at a concentration of20 μg/ml, in PB and RO water. Print buffer is printed as the negativecontrol in the assay. It indicates the overall assay background. Twentyeight μL of each of the prepared proteins and print buffer solution weretransferred in a 384-well PCR plate for printing. The printing wasperformed using the Thomas™ microarrayer under the following conditionstemperature—21.6° C. and relative humidity—40-43%. The pin is washed inRO water before each spot is printed. Visual quality control of theprinted array was performed to check spot positioning and morphology,before being sealed (parafilm) and incubated for ˜22 h at 2-8° C. . Twohundred μL of blocking solution was added to each well of the microtiterplate, and incubated at room temperature for 1 h. Plates were thenwashed three times with 300 μL, of washing solution. Any remainingliquid was removed, and plates were left to dry for 20 min at roomtemperature. The dried plates were sealed and stored at 2-8° C.

Plates were brought to room temperature prior to being used. One volumeof serum/plasma sample was added to 100 volumes of assay diluent (1:101dilution). Dilution ratios for anti-N Protein reconstructed human mAb,IgG and anti-Spike-RBD human reconstructed mAb, IgG were adjusted tomake a final concentration of 1 μg/ml for each Ab. The samples wereprepared in 1.1 ml tubes by mixing the added samples with the assaydiluent via multiple aspiration and dispensing runs. This was followedby the dispensing of 100 μL of each sample into the specific wells onthe microtiter plate. The plate was then sealed (parafilm) and incubatedfor 30 minutes at 37° C. , followed by washing the plate 3 times using300 μL of washing solution per well for each wash. Any excess liquid wasremoved by tapping the plate. One hundred μL of the secondary antibody(0.2 μg/ml) was added to each well of microtiter plate, which was thensealed (parafilm) and incubated for 15 minutes at 37° C. , followed byplate washing using 300 μL of washing solution per well for three washsteps. Any excess liquid was removed by inverting and tapping the plate.One hundred μL of Pierce 1-step ultra TMB Blotting solution was added toeach well of the microtiter plate, and incubated for 20 minutes at roomtemperature in the dark. The TMB was then removed by inverting andtapping the plate onto an absorbent tissue, removing any remainingliquid. Plate was read within 5 min. The plate was read using thesciREADER CL2, and the “R&D Imaging & Analysis” software. The camerafocus was set to 270 during the reading. The assay background wascalculated based on four chosen positions across a well.

The results showed that the method used to clean the pin was effective.The efficiency of the cleaning method is shown FIG. 10 where there areno colored spots appeared where the print buffer was printed.

The casein-based blocker used was very efficient in preventingnon-specific binding with-in wells while maintaining the spots'morphology. This is demonstrated in FIG. 11D-E, where a plasma samplereactive to both printed targets was compared to a non-reactive sample.For both samples, there is no excessive background in the well, for thereactive sample, as the assay spots are clearly differentiable from thebackground.

The signals obtained during the analysis of results, are automaticallyprocessed by the software, which calculates the mean of each spotintensity and subtracts the background (FIG. 10). The presence ofairborne particles at the bottom of the well occasionally increase thebackground signal, in these cases, an absolute value of 72AU was used asthe background median value. As each protein is printed in duplicate perwell, the median of the two replicates signal was used as the wellintensity. For the results presented, each sample was run in two wells,results are therefore an average of the four reactive spots (duplicatespots from two wells).

The ability of the assay to differentiate between anti-SARS-CoV-2Nucleocapsid Protein IgG antibodies and anti-SARS-CoV-2 SpikeGlycoprotein S1 IgG antibodies was tested using the Anti-N Proteinreconstructed human mAb, IgG (anti-N Protein mAb) and Anti-Spike-RBDhuman reconstructed mAb, IgG (Anti-Spike-RBD). When the anti-N ProteinmAb was used as the sample, only the assay spots printed with SARS-CoV-2Nucleocapsid Protein resulted in a positive reaction (FIG. 11B). On thecontrary, when the Anti-Spike-RBD mAb was used as the sample, only theassay spots with anti-SARS-CoV-2 SP reacted positively (FIG. 11C).

Two control samples were used to evaluate the performance of theCOVID-19 MIA. One of them was the Anti-SARS-CoV-2 Antibody, a samplecollected from a COVID-19 PCR positive-confirmed patient at least 4weeks after symptoms and recovery. This control showed good reactivityand a CV of less than 10% on both SARS-CoV-2 Nucleocapsid Protein andSARS-CoV-2 Spike Glycoprotein 51 targets (Table 1). The second controlmaterial was anti-SARS-CoV-2 QC1, a sample obtained from twoconvalescent plasma packs known to be SARS-CoV-2 positive. This samplehas shown a strong reactivity for SARS-CoV-2 NP but a low reactivity forSARS-CoV-2 SP, results gave a CV less than 5% for both NP and SP targets(Table 1).

TABLE 1 Assessment of some positive controls and the First WHOInternational Standard for Anti-SARS-CoV-2 IgG (human). SARS-CoV-2Nucleocapsid SARS-CoV-2 Spike Protein Protein as target (S1) as targetSamples tested Means CV Means CV Anti-SARS-CoV-2 Antibody 70.9 8% 31.3 8% Anti-SARS-CoV-2 QC1 58.2 1% 10.7  0% First WHO International 68.7 7%53.9 16% Standard for Anti-SARS-CoV-2 IgG

The assay was evaluated using the first WHO international standard foranti-SARS-CoV-2 immunoglobulin (human). The reactivities obtained forboth targets were high, however, the CV for the SARS-CoV-2 SpikeGlycoprotein S1 was approximately 16% (Table 1.). Even though theresults were promising, the assay was assessed using another referencepanel. The first WHO international reference panel for anti-SARS-CoV-2immunoglobulin, is made up of five referenced samples, which havedifferent reactivities against NP and SP. Using SARS-CoV-2 SpikeGlycoprotein S1 as the target, the assay signals for the reference panelcorrelated to the reactivity levels indicated by the provider, i.e., thesignal decreased from the highly reactive sample to the low reactiveone, and no reactivity for the negative sample (FIG. 12).

The performance of developed assay was compared with that of othercommercial tests using the samples in the NIBSC Anti-SARS-CoV-2verification panel. The panel comprises 37 samples, 23 samples fromconvalescent plasma packs known to be anti-SARS-CoV-2 positive and 14from convalescent plasma packs known to be anti-SARS-CoV-2 negative. Allsamples were tested using several commercial tests. The results obtainedare summarized in Table 2. As the developed assay can detect the IgGantibodies against SARS-CoV-2 Nucleocapsid Protein and IgG antibodiesagainst SARS-CoV-2 Spike Glycoprotein S1 in a single well, the resultsobtained with each target were compared to the commercial tests that usethe same target in their assay for NP and SP, i.e., SARS-CoV-2 NP andSARS-CoV-2 SP (S1, S 1/S2 or S1-RBD). Except for the Pictor assay, thevalues reported in the table are taken from the datasheet of NIBSCpanel. The values for the Pictor assay are the average of tworeplicates. Please note that the values are different for each company,which cannot be compared as they employ different readout mechanisms.

For both viral proteins, the 23 positive samples generate very strongsignals, which can clearly be differentiated from the negligible signalsobtained using the 14 negative samples. The results obtained correlatewith the results obtained by other commercial tests. For the SARS-CoV-2NP spots, the negative samples have a signal below 3AU (ArbitraryUnits). The only exception is panel #34, which has a signal of 5.8 thatis still very low considering the lowest signal obtained across thepositive ones is 52.3AU for panel#16. Whereas for SARS-CoV-2 SpikeGlycoprotein (S1) spots, there was no reactivity obtained on any of thenegative samples tested.

TABLE 2 Assessment of the Anti-SARS-CoV-2 NIBSC verification panel forserology assays. SARS-CoV-2 Nucleocapsid Protein as target SARS-CoV-2Spike Glycoprotein (S1) as target Abbott Euro- ROCHE Liaison SiemensEuro- Panel # Architect Immun Elecsys Pictor (S1/S2) (S1, RBD) ImmunPictor 1 3.7 2.7 17.2 85.9 20.2 0.6 1.7 34.9 2 1.4 2.1 8.8 65.6 37.50.98 3.2 46.2 3 6.5 6.2 50.7 85.3 260.7 >20.0 8.5 89.3 4 4.2 3.7 27.586.2 202 >20.0 7.9 88.2 5 7.2 5.8 90.5 67.0 226 >20.0 8.5 76.1 6 4.2 3.154.9 53.9 75 12.2 5.8 47.3 7 4 2.9 8.9 58.4 105.3 8.8 5.7 59.1 8 7.2 5101.3 67.2 163 >20.0 7.1 72.2 9 5.8 5.6 50.1 88.5 166.7 >20.0 7.9 90.010 5.8 5.5 49.9 84.6 174.3 >20.0 8 84.4 11 1.8 1.4 14.2 59.1 74.7 4.24.6 61.6 12 4.4 3.4 51 81.1 86.2 4.7 4.9 62.9 13 6.4 4.3 101.7 58.9 87.45.4 5.1 38.6 14 4.5 3.3 70.1 53.9 88.5 5.3 4.8 36.1 15 5.3 3.9 108 68.2110.7 6.5 5.7 51.9 16 1.2 2.4 4.6 52.3 79.1 1.9 4.2 34.8 17 3.9 3.1 26.691.7 111.7 12.2 6.1 73.9 18 6.4 4.9 82.5 87.6 161.3 >20.0 7.6 83.6 195.1 5.1 58.8 86.2 148 12.2 5.8 62.4 20 4.5 3.3 141.7 78.3 145.7 10.3 6.276.5 21 7 4.9 108.3 71.7 117.3 15.2 6.6 28.4 22 5.5 3.5 132 64.3 15113.5 6.7 54.7 23 5 3.4 123.3 65.1 140.7 8.4 6.5 57.8 24 0.02 0.05 0.020.0 <3.8 0 0.09 0.0 25 0.05 0.1 0.08 1.3 <3.8 0.03 0.08 0.4 26 0.12 0.170.07 2.9 <3.8 0.01 0.39 0.0 27 0.01 0.03 0.07 0.3 <3.8 0 0.08 0.0 280.05 0.03 0.07 0.0 <3.8 0 0.07 0.0 29 0.01 0.02 0.07 0.0 <3.8 0 0.06 0.030 0.16 0.15 0.08 0.0 <3.8 0.01 0.27 0.0 31 0.01 0.09 0.07 0.0 <3.8 00.1 0.0 32 0.03 0.04 0.07 0.0 <3.8 0 0.11 0.0 33 0.04 0.08 0.07 1.2 <3.80 0.11 0.3 34 0.01 0.18 0.08 5.8 <3.8 0 0.09 0.0 35 0.01 0.05 0.07 0.88.43 0 0.07 0.0 36 0.04 0.2 0.07 0.0 <3.8 0 0.07 0.0 37 0.03 0.06 0.070.0 <3.8 0 0.12 0.0

Example 22 COVID-19 MIA—Membrane Free, Single Well, With GroupedSARS-CoV-2 Structural Proteins

The printed spots in the COVID-19 MIA are shown as shaded circles whereeach circle of a particular shade corresponds to a specific SARS-CoV-2structural protein that is printed on the membrane or the solid surface(FIG. 13A-B). The white circles signify that nothing has been printed atthat specific position.

The plate preparation and spotting were performed as follows. One volumeof NP-40 0.05% was added to 9 volumes of antigens solution made ofSARS-CoV-2 NP & SARS-CoV-2 SP(S1). The preparation was incubated for 15min at room temperature. This is followed by the addition of 2X PB andthe required volume of RO water to the solution so that the targetantigen and print buffer concentrations are achieved. The final solutioncontained SARS-CoV-2 NP at 100 μg/ml and SARS-CoV-2 SP(S1) at 200 μg/mlin 1X PB. Biotinylated goat anti-mouse IgG is used as positive control.It is used to ascertain that the secondary antibody solution was addedto the well and helps the image analysis software to detect the printedarray. It was printed at a concentration of 20 μg/ml, which was achievedvia dilution in 2X PB and RO water. The print buffer is printed as suchon the array and is used as negative control for the assay. It indicatesthe quality of printing, the intensity of background induced by theprint buffer alone and, the overall assay background. Twenty eight μL ofeach of the prepared proteins and print buffer solution were transferredin a 384-well PCR plate for printing. The printing was performed usingthe Thomas™ microarrayer. The temperature inside the arrayer during theprinting was 22.8° C., while the humidity was between 40%. The array wasprinted in three steps: 1) spots A1, C1, E1 were printed first, which isfollowed sequentially by the printing of spots A2 & A3, and E2, E3 & E5.Before each spot is printed, the arrayer washes the pin with RO water inthe washing chamber and dries it in the drying chamber. The consecutivewash and dry steps are performed three times and then the pin goes inthe next source plate's well to collect the preparation and starts theprinting of respective spots. A visual quality control of the printedarray was done to ensure that the spots were at the right position andhad a good morphology. The plates were then sealed with parafilm andincubated for ˜19 h at 2-8° C. in a refrigerator. The blocker was storedbetween 2 to 8° C. (with an average temperature of 4° C.) since thepreparation day. It was left at room temperature for 25 min prior tobeing used, while the plate was left at the same temperature for 5 minprior to being blocked. Two hundred μL of blocking solution was added toeach well of the microtiter plate, which was then sealed using parafilmand incubated at room temperature for 1 h. This was followed by washingthe plate three times on a plate washer with 300 μL of washing solution.The washed plate was then tapped onto an absorbent tissue to remove anyremaining liquid and left in the biosafety cabinet for 20 min at roomtemperature. The dried plate was sealed and stored at 2-8° ° C. in arefrigerator.

The plate was left at room temperature for 30 min prior to being used.One volume of sample was added to 100 volumes of assay diluent (1:101dilution). However, appropriate dilution ratios were used for anti-NProtein reconstructed human mAb, IgG and anti-Spike-RBD humanreconstructed mAb, IgG to make a final concentration of 1 μg/ml for eachAb. The samples were prepared in 1.1 ml tubes by mixing the addedsamples with the assay diluent via multiple aspiration and dispensingruns. This was followed by the dispensing of 100 μL of each sample intothe specific wells on the microtiter plate. The plate was then sealedusing a parafilm and incubated for 30 minutes at 37° C. This wasfollowed by washing the plate 3 times using 300 μL of washing solutionper well for each wash. The washed plate was then tapped onto anabsorbent tissue to remove any remaining liquid. One hundred μL of thesecondary antibody (0.2 μg/ml) was added to each well of the microtiterplate, which was then sealed using a parafilm and incubated for 15minutes at 37° C. This was followed by washing the plate 3 times using300 μL of washing solution per well for each wash. The washed plate wasthen tapped onto an absorbent tissue to remove any remaining liquid. Onehundred μL of Pierce 1-step ultra TMB Blotting solution was added toeach well of the microtiter plate, which was then covered and incubatedfor 20 minutes at room temperature in the dark. The TMB was then removedby inverting the plate, which was tapped onto an absorbent tissue toremove any remaining liquid and read within 5 min. The plate was readusing the sciREADER CL2, and the “R&D Imaging & Analysis” section of thesoftware was used to read the plate. The camera focus was 270 during thereading. The assay background was calculated based on four chosenpositions across a well.

The results showed that the method used to clean the pin was effective.The efficiency of the cleaning method is shown FIG. 14 where there areno colored spots appeared where the print buffer was printed.

The casein-based blocker used was very efficient in protecting thenon-specific binding in wells while maintaining the spots' morphology.It is demonstrated in FIG. 15D-E where a plasma sample reactive to bothprinted targets and a non-reactive one was tested. For both samples,there is no excessive background in the well and for the reactivesample, the assay spots are clearly differentiable from the background.

The signal used during the analysis are automatically processed by thesoftware, which calculates the mean of each spot intensity and subtractsthe background (FIG. 14). The presence of some airborne particles at thebottom of the well may increase the background signal. For those cases,an absolute value of 72AU was used as the background median value. Aseach protein is printed in duplicate per well, the median of the tworeplicates signal was used as the well intensity. Each sample was run intwo wells. Therefore, the values reported here are the summary of thoseduplicates.

To demonstrate the capability of the platform to give a signal if thesample is only positive for anti-SARS-CoV-2 Nucleocapsid Protein IgGantibodies or anti-SARS-CoV-2 Spike Glycoprotein S1 IgG antibodies, itwas tested against the following monoclonal antibodies: Anti-N Proteinreconstructed human mAb, IgG (anti-N Protein mAb) and Anti-Spike-RBDhuman reconstructed mAb, IgG (Anti-Spike-RBD). The assay spots werevisible both when anti-N Protein mAb (FIG. 15B) and Anti-Spike-RBD mAbwere used as samples (FIG. 15C).

Two control samples were used. The Anti-SARS-CoV-2 Antibody gave goodresults with a CV of less than 10% (Table 3). Similarly, anti-SARS-CoV-2QC1 also showed good results with a CV of around 12% (Table 3).

TABLE 3 Assessment of some positive controls and the First WHOInternational Standard for Anti-SARS-CoV-2 IgG (human) SARS-CoV-2Nucleocapsid Protein & Spike (S1) as target Samples tested Means CVAnti-SARS-CoV-2 Antibody 66.6  8% Anti-SARS-CoV-2 QC1 29.3 12% First WHOInternational Standard for Anti- 71.9 12% SARS-CoV-2 IgG

The developed assay was evaluated using the first WHO internationalstandard for anti-SARS-CoV-2 immunoglobulin (human), which showed goodresults with a CV of about 12% (Table 3). Further, the first WHOinternational reference panel for anti-SARS-CoV-2 immunoglobulin wasalso tested. The assay signals correlated to the levels indicated by theprovider, i.e., the signals decrease from the high reactive panel sampleto the low reactive one and no signal for the negative sample. However,it is important to take into consideration the standard deviation ofsamples (FIG. 16).

The performance of the developed assay was compared with that of othercommercial tests using the samples in the Anti-SARS-CoV-2 verificationpanel. The panel comprises 37 samples, which has twenty-three samplesfrom convalescent plasma packs known to be anti-SARS-CoV-2 positive andfourteen from convalescent plasma packs known to be anti-SARS-CoV-2negative. All samples were tested using several commercial tests. Theresults obtained are summarized in Table 4. As the developed assay candetect the IgG antibodies against SARS-CoV-2 Nucleocapsid Protein andIgG antibodies against SARS-CoV-2 Spike Glycoprotein S1 in a singlewell, the results obtained with each target were compared to thecommercial tests that use the same target in their assay for NP and SP,i.e., SARS-CoV-2 NP and SARS-CoV-2 SP (S1, S1/S2 or S1-RBD).

For both viral proteins, the twenty-three positive samples generate verystrong signals, which can clearly be differentiated from the negligiblesignals obtained using the fourteen negative samples. The resultsobtained correlate with the results obtained by other commercial tests.The negative samples had signals of less than 1AU.

Except for Pictor, the values reported in the table are taken from thedatasheet of NIBSC panel. The values for Pictor are the average of tworeplicates. Please note that the values are different for each company,which cannot be compared as they employ different readout mechanisms.

TABLE 4 Assessment of the Anti-SARS-CoV-2 verification panel forserology assays. SARS-CoV-2 Nucleocapsid SARS-CoV-2 Spike Pan- Proteinas target Glycoprotein (S1) as target el Abbott Euro- ROCHE Euro-Liaison Siemens Pic- # Architect Immun Elecsys Immun (S1/S2) (S1, RBD)tor 1 3.7 2.7 17.2 1.7 20.2 0.6 87.6 2 1.4 2.1 8.8 3.2 37.5 1.0 70.8 36.5 6.2 50.7 8.5 260.7 >20.0 86.5 4 4.2 3.7 27.5 7.9 202.0 >20.0 87.2 57.2 5.8 90.5 8.5 226.0 >20.0 72.4 6 4.2 3.1 54.9 5.8 75.0 12.2 58.0 74.0 2.9 8.9 5.7 105.3 8.8 67.3 8 7.2 5.0 101.3 7.1 163.0 >20.0 76.1 95.8 5.6 50.1 7.9 166.7 >20.0 96.9 10 5.8 5.5 49.9 8 174.3 >20.0 91.6 111.8 1.4 14.2 4.6 74.7 4.2 68.4 12 4.4 3.4 51.0 4.9 86.2 4.7 76.7 13 6.44.3 101.7 5.1 87.4 5.4 66.7 14 4.5 3.3 70.1 4.8 88.5 5.3 60.9 15 5.3 3.9108.0 5.7 110.7 6.5 77.9 16 1.2 2.4 4.6 4.2 79.1 1.9 61.6 17 3.9 3.126.6 6.1 111.7 12.2 94.1 18 6.4 4.9 82.5 7.6 161.3 >20.0 89.8 19 5.1 5.158.8 5.8 148.0 12.2 78.8 20 4.5 3.3 141.7 6.2 145.7 10.3 71.9 21 7.0 4.9108.3 6.6 117.3 15.2 71.9 22 5.5 3.5 132.0 6.7 151.0 13.5 66.4 23 5.03.4 123.3 6.5 140.7 8.4 71.2 24 0.0 0.1 0.0 0.09 <3.8 0.0 0.0 25 0.1 0.10.1 0.08 <3.8 0.0 0.0 26 0.1 0.2 0.1 0.39 <3.8 0.0 0.0 27 0.0 0.0 0.10.08 <3.8 0.0 0.0 28 0.1 0.0 0.1 0.07 <3.8 0.0 0.0 29 0.0 0.0 0.1 0.06<3.8 0.0 0.0 30 0.2 0.2 0.1 0.27 <3.8 0.0 0.0 31 0.0 0.1 0.1 0.1 <3.80.0 0.0 32 0.0 0.0 0.1 0.11 <3.8 0.0 0.0 33 0.0 0.1 0.1 0.11 <3.8 0.00.0 34 0.0 0.2 0.1 0.09 <3.8 0.0 0.0 35 0.0 0.1 0.1 0.07 8.4 0.0 0.0 360.0 0.2 0.1 0.07 <3.8 0.0 0.0 37 0.0 0.1 0.1 0.12 <3.8 0.0 0.0

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A substrate comprising at least two capture elements specific forSARS-CoV-2 on the substrate, each capture element corresponding to andbeing able to bind a target analyte, the substrate further optionallycomprising a plurality of control elements comprising: a) at least onefiduciary marker, b) at least one negative control to monitor backgroundsignal, c) at least one negative control to monitor assay specificity,d) at least one positive colorimetric control, e) at least one positivecontrol to monitor assay performance and any combination thereof.
 2. Thesubstrate of claim 1, wherein the capture elements bind target analytes,wherein the target analytes are indicative of antibodies produced inresponse to SARS-CoV-2 infection.
 3. The substrate of claim 1, whereinthe capture elements are selected from a protein, a protein fragment, apeptide, a polypeptide, a polypeptide fragment, an antibody, an antibodyfragment, an antibody binding domain or any combination thereof.
 4. Thesubstrate of claim 3, wherein the capture elements are selected from aSARS-CoV-2 Membrane protein (MP), Nucleocapsid protein (NP), Spikeprotein (SP), or any combination thereof
 5. The substrate of claims 3,wherein the target analyte is a SARS-CoV-2 antibody, fragment or bindingdomain thereof.
 6. The substrate of claim 1, wherein the target analyteis selected from a protein, a protein fragment, an antigen, an antigenicdeterminant, an epitope, a hapten, an immunogen, an immunogen fragment,a virus protein, or any combination thereof.
 7. The substrate of claim6, wherein the capture element is a virus structural protein or epitopethereof.
 8. The substrate of claim 7, wherein the virus structuralprotein or epitope thereof is selected from a SARS-CoV-2 Membraneprotein (MP), Nucleocapsid protein (NP), Spike protein (SP), fragmentsthereof or any combination thereof.
 9. The substrate of claim 7, whereinthe virus structural protein or epitope thereof is aNucleocapsid/spike/membrane protein or fragment thereof.
 10. Thesubstrate of claim 1, wherein the substrate is a solid or poroussubstrate.
 11. The substrate of claim 10, wherein the solid substrate isa paramagnetic bead, microtiter plate, microparticle, or a magneticbead.
 12. The substrate of claim 10, wherein the porous substrate is amembrane.
 13. A kit for detecting a plurality of target analytes in asample, comprising a) a substrate of claim 1 and optionally one or bothof b) a background reducing reagent, and c) a colorimetric detectionsystem.
 14. The kit of claim 13, further comprising one or more itemsselected from the group consisting of: a) a wash solution, b) one ormore antibodies for detection of antigens, ligands or antibodies boundto the capture elements or for detection of the positive controls, c)software for analyzing captured target analytes, and d) a protocol formeasuring the presence of target analytes in samples.
 15. The kit ofclaim 14, wherein the antibodies for detection comprise antibody-bindingprotein (BP) conjugates, antibody-enzyme label conjugates, or anycombination thereof.
 16. The kit of claim 14, wherein the sample is ablood sample.
 17. The kit of claim 16, wherein the blood sample is serumor plasma.
 18. The kit of claim 13, wherein the substrate is a solid orporous substrate.
 19. The kit of claim 18, wherein the solid substrateis a paramagnetic bead, microtiter plate, or microparticle.
 20. The kitof claim 18, wherein the porous substrate is a membrane.
 21. A method ofdetecting antibodies produced in a subject in response to SARS-CoV-2infection comprising: contacting a substrate of claim 1 with abiological sample from the subject, wherein the subject is suspected ofhaving COVID-19 or is exposed to SARS-CoV-2; and detecting the presenceof an antibody produced in response to SARS-CoV-2 infection.
 22. Themethod of claim 21, wherein the detection method is a colorimetric,absorbance, chemiluminescence or a fluorescence signal.
 23. The methodof claim 21, wherein the detection method is electrochemical, surfaceplasmon resonance, localized surface plasmon resonance orinterferometry.
 24. The method of claim 21, wherein the antibody is IgG.25. The method of claim 21, wherein the sample is a blood sample. 26.The method of claim 27, wherein the blood sample is serum or plasma. 27.A method for processing a microarray comprising: a) providing asubstrate of claim 1; b) adding at least one sample to the substrate;and c) processing the substrate such that a detectable result is givenby two or more of i) at least one fiduciary marker, ii) at least onepositive colorimetric control, and iii) at least one positive control tomonitor assay performance.
 28. A method for detecting an analyte in asample comprising providing a substrate of claim 1, adding at least onesample to the substrate, and processing the substrate such that adetectable result is provided.
 29. The method of claim 28, wherein thedetectable result includes two or more of at least one fiduciary marker,at least one positive colorimetric control, and at least one positivecontrol to detect an analyte in the sample.